1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to plant genes that activate wax biosynthesis and methods of use thereof.
2. Description of the Related Art
A cuticle layer covers most primary aerial organs of vascular plants and forms the contact zone between the plant and the environment (Kerstiens, 1996). Cuticular waxes are the major constituents of plant cuticle and play an important role in protecting aerial organs from damage caused by environmental stresses. Cuticular waxes are complex mixtures of very long chain fatty acids, alkanes, primary and/or secondary alcohols, aldehydes, ketones, esters, triterpenes, sterols, and flavonoids. Wax compounds can be embedded within the cutin polymer framework and form intracuticular wax. In many plants, however, more waxes are loaded outside of the cuticle membrane and form an epicuticular wax layer that give the plant surface a glaucous or grey appearance (Jenks and Ashworth, 1999; Post-Beittenmiller, 1996).
Plant cuticular wax biosynthesis and its loading to the plant surface is a complicated but actively regulated process (Broun et al., 2004; Jenks et al., 2002). Mutant analysis has contributed to the identification of some of the components and genes involved in wax deposition. Mutants with reduced wax accumulation or altered wax composition are in general characterized by a bright green phenotype which can be detected visually (Aarts et al., 1995). In Arabidopsis, 120 cuticular wax mutants representing a total of 31 recessive mutant loci have been identified, although dominant wax gene mutations have not been reported (Jenks et al., 2002). Wax-deficient mutants have also been identified in other species, including maize, sorghum, barley and rape (Kunst and Samuels, 2003).
Studies on the eceriferum (cer) mutants and T-DNA insertional mutants in Arabidopsis and glossy (gl) mutants in maize led to the identification and isolation of a number of wax-related genes. To date, 12 genes associated with wax production or regulation have been identified by molecular-genetic approaches. Among these genes, CER1, CER2, CER6/CUT1, 3-ketoacyl-CoA synthase (KCS1), FIDDLEHEAD (FDH), GL1, GL8 and WAX2, may encode metabolic enzymes or be involved in transport of wax compounds (Aarts et al., 1995; Chen et al., 2003; Fiebig et al., 2000; Hansen et al., 1997; Millar et al., 1999; Negruk et al., 1996; Pruitt et al., 2000; St-Pierre et al., 1998; Todd et al., 1999; Xia et al., 1996, 1997; Xu et al., 1997), while CER3, GL2, GL15 and WIN1/SHINE1 appear to encode regulatory proteins (Aharoni et al., 2004; Broun et al., 2004; Hannoufa et al., 1996; Moose and Sisco, 1996; Tacke et al., 1995). Mutations in most of these genes showed altered wax accumulation (Jenks et al., 2002), cosuppression of some of the genes resulted in waxless stems in Arabidopsis (Millar et al., 1999; Todd et al., 1999), and overexpression of some of the genes in Arabidopsis mutant background complemented corresponding mutant phenotypes (Fiebig et al., 2000; Hannoufa et al., 1996). However, only limited information is available on the effects of overexpression of these genes in a wild-type background.
Overexpression of the condensing enzyme gene CER6/CUT1 under the control of CaMV35S promoter failed to promote wax deposition (Millar et al., 1999), while under the control of epidermis-specific CER6 promoter, CER6/CUT1 overexpression led to increased wax load in stems of Arabidopsis (Hooker et al., 2002). The only report of increased wax accumulation in leaf tissues of Arabidopsis was by the overexpression of a transcriptional activator (Broun et al., 2004; Aharoni et al., 2004).
Transcription factors are regulatory proteins that modulate gene expression through sequence-specific DNA binding and/or protein-protein interactions. They are capable of activating or repressing transcription of target genes as switches of the regulatory cascade. Most of the transcription factors are grouped into gene families according to their well-conserved DNA-binding domains.
APETALA 2 (AP2)/Ethylene-responsive element binding factors (ERF or EREBP) domain-containing transcription factor is a group of transcriptional regulators that are specifically found in plants (Okamuro et al., 1997; Riechmann et al., 2000). The AP2 domains in these proteins play a major role in specific promoter DNA sequence/element binding and transcriptional activation (Okamuro et al., 1997; Sakuma et al., 2002). This gene family has been further grouped into three major subfamilies and some smaller groups based on their functions and sequence similarities (Dubouzet et al., 2003; Riechmann et al., 2000). The AP2 subfamily genes (containing double AP2 domains) were thought to developmentally control flowering time in plants (Jofuku et al., 1994; Schultz and Haughn, 1991). The genes in ERF subfamily have been found to be involved in plant response to pathogen infection and mediate disease resistance (Chakravarthy et al., 2003; Gutterson and Reuber, 2004; Onate-Sanchez and Singh, 2002).
Recently, a new group of AP2 domain-containing transcription factors, dehydration-response element binding protein (DREB)/C-repeat binding factor (CBF), have been identified and characterized (Novillo et al., 2004; Shinozaki et al., 2003; Thomashow, 1999). They are mainly involved in the regulation of abiotic stress inducible genes; overexpression of some members from this subfamily in transgenic Arabidopsis induced a host of genes and conferred stress tolerance (Gilmour et al., 2000; Haake et al., 2002; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Stockinger et al., 1997).
While the foregoing studies have provided a further understanding of the metabolism of wax in plants, the prior art has generally failed to provide wax biosynthesis activating genes, for example, that would yield drought tolerance. The identification of such genes would allow the creation of novel plants with improved phenotypes and methods for use thereof. There is, therefore, a great need in the art for the identification of plant genes that activate wax biosynthesis and methods for their use.